ABSTRACT The relatively recent development of droplet-based single cell RNA sequencing (scRNA-seq) techniques has changed the landscape of several fields of research. It has enabled investigators to elucidate the cellular complexities in tissues, organs, and tumors which have traditionally been treated as homogeneous. The new perspective is important for understanding the biology underlying normal and disease conditions, and it will stimulate development of new therapies that take advantage of the more granular understanding of physiological systems. With this new perspective comes a realization of some of the limits to scRNA-seq, and barriers that must be overcome to achieve potential of single cell analyses. This proposal focuses on developing an innovative new technology that will dramatically improve the impact of scRNA- seq techniques applied to stem cell research and regenerative medicine therapies. Currently, scRNA-seq is great at identifying the cellular components within a sample, but it is incapable of discerning the route individual cells took through a cell lineage. It is critical to determine cell lineages because they provide the essential instructions informing protocols for directed differentiation and cell based therapies. The proposal's system, named Cell Lineage Annotating DNA Elements (CLADE) will introduce into stem cells a library of individually barcoded plasmid DNAs. Using elements of episomal DNA maintenance system from the Epstein-Barr virus, the CLADE plasmid barcode will change as cells undergo cell divisions, resulting in a unique cohort of barcoded plasmids for individual cells. By using the same reaction to generate scRNA-seq transcriptomes and CLADE identification, the system will provide a seamless way of determining ancestry of cells alongside their transcriptomic identity. Through a series of six Milestones, the CLADE system will be synthesized, tested, and developed so that it can be readily used by other groups using scRNA-seq and stem cells. We anticipate CLADE will enable a near comprehensive cellular ancestry to be determined for >40,000 individual cells for >10 generations of cell division. Compared to current capabilities of hundreds of cells and two or three cell divisions, CLADE can massively increase capacity for identifying cell lineages and have a long lasting impact on fields that benefit from scRNA-seq research.